The present invention relates to neuronal progenitor cells which have been identified in both tissue cultures and histological sections of the adult human brain. The present invention provides methods for the localization, characterization, harvest, and propagation of neuronal progenitor cells derived from adult humans.
The damaged adult mammalian brain is incapable of significant structural self-repair. Terminally differentiated neurons are incapable of mitosis, and compensatory neuronal production has not been observed in any mammalian models of structural brain damage (Korr, 1980; Sturrock, 1982). Although varying degrees of recovery from injury are possible, this is largely because of synaptic and functional plasticity rather than the frank regeneration of neural tissues. The lack of structural plasticity of the adult brain is partly because of its inability to generate new neurons, a limitation that has severely hindered the development of therapies for neurological injury or degeneration. Indeed, the inability to replace or regenerate damaged or dead cells continues to plague neuroscientists, neurologists, and neurosurgeons who are interested in treating the injured brain. During the last several years, however, a considerable body of evidence has evolved that suggests a marked degree of cellular plasticity in the adult as well as in the developing CNS. In particular, recent work on neural progenitor cells, derived from both embryos and adults, has suggested strategies for directed neuronal regeneration and structural brain repair. These include the use of neural stem cells which are the multipotential progenitors of neurons and glia that are capable of self-renewal (Davis, 1994; Gritti, 1996; Kilpatrick, 1993; Morshead, 1994; Stemple, 1992; Goldman, 1996; Weiss, 1996a).
In the adult human brain, both neuronal and oligodendroglial precursors have been identified as well, and methods for their harvest and enrichment have been established. Neural precursors have several characteristics that make them ideal vectors for brain repair. They may be expanded in tissue culture, providing a renewable supply of material for transplantation. Moreover, progenitors are ideal for genetic manipulation and may be engineered to express exogenous genes for neurotransmitters, neurotrophic factors, and metabolic enzymes (reviewed in Goldman 1998; Pincus 1998; and Goldman and Luskin 1998).
In embryonic neurogenesis, the proliferation of neuronal precursors takes place at the surface of the central canal lining the neural tube (Jacobson, 1991). The central canal ultimately forms the ventricular system of the adult. This neurogenic layer is referred to as the ventricular/subventricular zone in development, and the ependymal/subependymal zone (SZ) in adults (Boulder Committee, (1970). In development, mitogenesis in the ventricular/subventricular zone is followed by the migration of newly generated neurons and glia along radial guide fibers into the brain parenchyma, including that of the cortical plate (LaVail, 1971; Rakic, 1971; Rakic, 1974; Sidman, 1973).
A variety of signals, including both humoral and contact-mediated factors, have been described which influence the proliferation, differentiation, and survival of stem cells and their progeny. Work on model systems derived from the peripheral nervous system has suggested that the neurotrophins (Anderson, 1986; DiCicco-Bloom, 1993; Murphy, 1991; Sieber-Blum, 1991), neurotransmitters (Pincus, 1990), and traditional growth factors (DiCicco-Bloom, 1988; Murphy, 1994; Shah, 1994) may all influence the development of precursors in vitro. In the CNS, soluble growth factors, particularly basic fibroblast growth factor (FGF-2) regulate neuronal precursor proliferation (DeHamer, 1994; Deloulme, 1991; Drago, 1991; Gensburger, 1987; Gritti, 1996; Kilpatrick, 1995; Kitchens, 1994; Murphy, 1990; Palmer, 1995; Qian, 1997; Ray, 1994; Ray, 1993; Vescovi, 1993). In mixed cell cultures derived from rat embryonic cerebrum, the addition of FGF-2 stimulated the proliferation of neuronal precursors (Gensburger, 1987). Similarly, FGF-2 stimulated the proliferation of a multipotential neural progenitor in fetal mice, which gave rise to neurons and astrocytes (Kilpatrick, 1995). Embryonic rat hippocampal, spinal cord, and olfactory neuron progenitors all have been shown to proliferate in the presence of FGF-2 (DeHamer, 1994; Deloulme, 1991; Ray, 1994; Ray, 1993). Not surprisingly, FGF-2 may also regulate precursor division in concert with other factors; this has been demonstrated in the coordinate regulation of neuronal precursor division by insulin-like growth factor I and FGF-2 (Drago, 1991), as well as oligodendrocyte precursor division by FGF-2 and platelet-derived growth factor (McKinnon, 1993; Wolswijk, 1992).
Where FGF2 had been shown to promote the division of neuronal precursor cells and, hence, the specific generation of neurons, epidermal growth factor (EGF) has also been shown to influence the proliferation of uncommitted neural precursors (Kitchens, 1994; Lu, 1996; Ray, 1994; Reynolds, 1992b; Reynolds, 1992a; Santa-Olalla, 1995; Weiss, 1996b). In dissociated cultures of embryonic mouse striata grown in suspension without culture substrata, EGF induced the proliferation of progenitor cells and the formation of floating xe2x80x9cneurospheresxe2x80x9d of cells, which expressed nestin (Reynolds, 1992a). Nestin is an intermediate filament protein expressed not only by CNS stem cells (Dahlstrand, 1992; Lendahl, 1990a), but also by young neurons reactive astrocytes, and radial glia. When these neurospheres were dissociated and plated onto poly-L-ornithine-coated plates, xcex3-aminobutyric acid- and substance P-expressing neurons and glial fibrillary acidic protein-expressing astrocytes were generated (Ahmed, 1995). Similar effects were reported in adult striatal cultures (Reynolds, 1992b). In this culture preparation, the actions of EGF were mimicked by its membrane-bound homolog, transforming growth factor xcex1, but not by nerve growth factor, FGF-2, platelet-derived growth factor, or transforming growth factor xcex2. A similar action of EGF on precursor cells derived from embryonic and adult rat spinal cord has also been reported (Ray, 1994; Weiss, 1996).
Although it is now possible to isolate and cultivate populations of neural precursors in vitro, the ability to direct specific neuronal phenotypes has remained elusive. In the EGF-generated sphere model, multipotent progenitors differentiated into neurons, which expressed xcex3-aminobutyric acid and substance P, as well as astrocytes and oligodendrocytes (Reynolds, 1992b; Reynolds, 1992a; Vescovi, 1993; Weiss, 1996b). Other neuronal phenotypes were rare, and their directed differentiation into defined transmitter phenotypes has not yet been demonstrated. In this regard, Raff et al. (Raff, 1988; Raff, 1983) suggested that growth factors control the development of a bipotential glial progenitor. Sequential exposure to specific combinations of platelet-derived growth factor, ciliary neurotrophic factor, and neurotrophin 3 can direct clonal expansion of the oligodendrocyte/Type 2 astrocyte (02A) progenitor cell in vitro, and drive an intrinsic clock that times oligodendrocyte development (Barres, 1994; Lillien, 1988; Raff, 1988; Temple, 1985). Nonetheless, a similarly directed differentiation of multipotent stem cells along specific neuronal lines has not yet been clearly demonstrated.
The persistence of neuronal precursors in the adult mammalian brain may permit the design of novel and effective strategies for central nervous system repair. However, although methods for the characterization and propagation of progenitors derived from adult rodents have been described, no such methods have allowed the high-yield harvest of purified native progenitors. Furthermore, no methods have been reported for obtaining or propagating such progenitor cells from adult human brain tissue.
The present invention provides human neural or neuronal progenitor cells isolated and enriched from non-embryonal brain tissue of a human.
Another aspect of the present invention is a method of propagating neurons from progenitor cells derived from brain tissue by serially applying FGF2 and BDNF to the cells.
The present invention also provides a method of treating neurological damage by transplanting or implanting neuronal progenitor cells into the brain of a human patient. Human neuronal progenitor cells isolated from an adult human using the methods of the present invention are transplanted or implanted into the brain of a patient.
Yet another aspect of the invention is a method of enhancing the survival and function of neural or neuronal precursor cells or the cells descended from the neural or neuronal precursor cells by transducing the neural or neuronal precursor cells with a gene encoding an autocrine neurotrophin or an adhesion molecule.
A further embodiment of the invention is a method of treating a patient with a neurological disease resulting from the loss of expression or mutation of a gene required for neuronal function. A gene which encodes a functional protein which complements the loss of expression or mutation of the gene required for neuronal function is transfected into postnatal or adult human neuronal progenitor cells. The neuronal progenitor cells are then introduced into the brain of the patient.
The present invention also provides a method of detecting neural or neuronal progenitor cells. An antibody, which is directed against an RNA binding protein that is selectively and specifically expressed by neural or neuronal progenitor cells when compared to other cell types, is contacted with cells and those cells which are bound by the antibody are detected.
The present invention also relates to a method of separating human, non-embryonal neural or neuronal progenitor cells from a mixed population of cells from human brain tissue. In accordance with this method, a promoter which functions only in the human postnatal neural and neuronal progenitor cells is selected. A nucleic acid molecule encoding a fluorescent protein, under control of said promoter, is then introduced into the mixed population of cells. The non-embryonal neural or neuronal progenitor cells thereof are allowed to express the fluorescent protein. The fluorescent cells are separated from the mixed population of cells, where the separated cells are the neural or neuronal progenitor cells.
Yet another embodiment of the invention is a method of expressing a gene in the brain of a patient. Adult human neuronal progenitor cells are isolated. The cells are transformed with a gene and are then transplanted into the brain of a patient. The gene is then expressed in the brain of the patient.
Prior to this invention, a method for progenitor cells were only available from embryos. However, this source is problematic due to legal and ethical concerns resulting from the harvesting of embryonic tissue. Furthermore, the embryonic tissue may not be immunologically compatible with the patient""s tissue. The present invention provides a method for isolating and propagating progenitor cells isolated from the brain tissue of an adult human. The isolated progenitors can then be propagated in vitro or in vivo to treat nervous system damage. This method provides a non-embryonic source of progenitor cells, that may be derived from the patient""s own tissue.